Altering levels of anti-nutrient factors in plants

Abstract

A method for reducing the level of one or more than one protein in a plant or a tissue within the plant, and plants with reduced levels of target proteins are provided. The method involves introducing a nucleic acid sequence into the plant, where the nucleic acid sequence comprises a regulatory region operatively associated with a silencing nucleotide sequence. The expression of the silencing nucleotide sequence reduces or eliminates the expression of two or more than two enzymes involved in the synthesis of the one or more than one protein. The reduced level of the one or more than one protein may be determine by comparing the level of the one or more than one protein in the plant, or a tissue of the plant, with a level of the one ore more than one protein in a second plant, or the tissue from the second plant, that does not express the nucleic acid sequence. Plants and seeds obtained from plants with reduced levels of target proteins, and reduced anti-nutrient factors are also provided

1 . A method for reducing the level of one or more than one protein in a plant or a tissue within the plant comprising, 
 i) introducing a nucleic acid sequence into the plant, the nucleic acid sequence comprising a regulatory region operatively associated with a silencing nucleotide sequence, wherein expression of the silencing nucleotide sequence reduces or eliminates the expression of two or more than two enzymes involved in the synthesis of the one or more than one protein, and    ii) expressing the silencing nucleotide sequence within the plant or a tissue within the plant, to reduce the level of the one or more than one protein in the plant or within a tissue of the plant, the reduced level of the one or more than one protein is determine by comparing the level of the one or more than one protein in the plant, or a tissue of the plant, with a level of the one ore more than one protein in a second plant, or the tissue from the second plant, that does not express the silencing nucleic acid sequence.    
     
     
         2 . The method of  claim 1  wherein the regulatory region is selected from the group consisting of a constitutive regulatory region, an inducible regulatory region, a developmentally regulated regulatory region, and a tissue specific regulatory region.  
     
     
         3 . The method of  claim 2 , wherein the regulatory region is a tissues specific regulatory region.  
     
     
         4 . The method of  claim 1  wherein the level of the one or more than one protein is reduced by about 25 to about 100%, where compared to the level of the same one or more than one protein obtained from second plant.  
     
     
         5 . The method of  claim 1 , wherein the silencing nucleotide sequence is selected from the group consisting of an antisense RNA encoding nucleotide sequence, a ribozyme encoding sequence, and an RNAi encoding nucleotide sequence.  
     
     
         6 . The method of  claim 5 , wherein the silencing nucleotide sequence is a gene fusion.  
     
     
         7 . The method of  claim 6 , wherein the gene fusion comprises nucleic acid sequences encoding from two to ten gene sequences.  
     
     
         8 . The method of  claim 1  wherein the protein is involved in the synthesis of an anti-nutrient factor in seed tissue.  
     
     
         9 . The method of  claim 8 , wherein the anti-nutrient factor is selected from the group consisting of sinapine, phytate, fiber, and lignin.  
     
     
         10 . The method of  claim 8 , wherein the two or more than two enzymes are involved in a pathway of phenylpropanoid biosynthesis leading to sinapine synthesis, a pathway of phenylpropanoid biosynthesis leading to lignin synthesis, a pathway of phytate biosynthesis, or a combination thereof.  
     
     
         11 . The method of  claim 9  wherein the anti-nutrient factor is sinapine, and the two or more than two enzymes are involved in a pathway of phenylpropanoid biosynthesis.  
     
     
         12 . The method of  claim 11 , wherein the two or more than two enzymes are selected from the group consisting of phenylalanine ammonia lyase (PAL), cinnamate 4 hydroxylase (C4H), coumarate 3 hydroxylase (C3H), O-methyl transferase (OMT), ferulic acid hydroxylase (FAH), sinapate: UDP-glucose sinapoyltransferase (SGT), sinapolyglucose:choline sinapoyltransferase (SCT), S-adenosylmethionine synthase (SAMS),  
     
     
         13 . The method of  claim 12 , wherein the two or more than two enzymes are FAH and SCT.  
     
     
         14 . The method of  claim 12 , wherein the silencing nucleotide sequence is selected from the group consisting of an antisense RNA nucleotide sequence encoding sequence, a ribozyme encoding sequence, and an RNAi encoding nucleotide sequence.  
     
     
         15 . The method of  claim 9  wherein the anti-nutrient factor is lignin, and the two or more than two enzymes are involved in a pathway of phenylpropanoid biosynthesis.  
     
     
         16 . The method of  claim 9  wherein the anti-nutrient factor is lignin, and the nucleotide sequence encodes two or more proteins selected from the group consisting of cinnamic acid 4 hydroxylase (C4H), coumaric acid 3 hydroxylase (C3H), caffeic acid O-methyl transferase (COMT), ferulic acid hydroxylase (FAH), S-adenosylmethionine synthase (SAMS), 4-coumarate:CoA lyase (4CL), cinnamoyl-CoA reductase (CCR), phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD).  
     
     
         17 . The method of  claim 16 , wherein the two or more than two enzymes are selected from the group consisting of FAH-COMT, C3H-C4H, and 4CL-CCR.  
     
     
         18 . The method of  claim 17 , wherein the silencing nucleotide sequence is selected from the group consisting of an antisense RNA nucleotide sequence encoding sequence, a ribozyme encoding sequence, and an RNAi encoding nucleotide sequence.  
     
     
         19 . The method of  claim 9  wherein the anti-nutrient factor is phytate, and the two or more than two enzymes are involved in a pathway of phytate biosynthesis.  
     
     
         20 . The method of  claim 9  wherein the anti-nutrient factor is phytate, and the nucleotide sequence encodes two or more proteins selected from the group consisting of inositol 1,3,4-trisphosphate 5/6-kinase (IP3K), myo-inositol hexaphosphate kinase (IP6K), 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase (PIBP PDE), phosphatidylinositol phophatidylcholine transfer protein (PI/PC TP), inositol polyphosphate 5-phosphatase II (IPP), phosphatidylinositol-4-phosphate 5-kinase (PIKa), CDP-diacylglycerol-inositol 3-phosphatidyltransferase (phosphatidylinositol synthase, PIS), inositol polyphosphate 6-/3-/5-kinase 2b (IPK2a & IPK2b), inositol polyphosphate 5′-phosphatase I (IPP), myo-inositol-1-phosphate synthase (MIP), phosphatidylinositol kinase (PIPK), phosphatidylinositol 3-kinase (PI3K), myo-inositol monophosphatase (MIM), and phosphatidylinositol 3- and 4-kinase (PIKb).  
     
     
         21 . The method of  claim 21 , wherein the two or more than two enzymes are selected from the group consisting of IP3K-IP6K and PIKa-PIKb.  
     
     
         22 . The method of  claim 21 , wherein the silencing nucleotide sequence is selected from the group consisting of an antisense RNA nucleotide sequence encoding sequence, a ribozyme encoding sequence, and an RNAi encoding nucleotide sequence.  
     
     
         23 . A construct comprising a silencing nucleotide sequence, the silencing nucleotide sequence encoding two or more than two sequences that reduce or inhibit the expression of two or more that two enzymes involved in the synthesis of the one or more than one protein leading to sinapine biosynthesis, lignin biosynthesis, phytate biosynthesis, or a combination thereof.  
     
     
         24 . A plant comprising a nucleic acid sequence comprising a regulatory region operatively associated with a silencing nucleotide sequence, the silencing nucleotide sequence reducing or eliminating the expression of two or more than two enzymes involved in the synthesis of the one or more than one protein leading to sinapine biosynthesis, lignin biosynthesis, phytate biosynthesis, or a combination thereof.  
     
     
         25 . A method for reducing the level of one or more than one protein within a plant or a tissue within the plant comprising, expressing a nucleotide sequence within the plant or a tissue within the plant, the nucleic acid sequence comprising a regulatory region operatively associated with a silencing nucleotide sequence, where expression of the silencing nucleotide sequence reduces or eliminates the expression of two or more than two enzymes involved in the synthesis of the one or more than one protein leading to sinapine biosynthesis, lignin biosynthesis, phytate biosynthesis, or a combination thereof, the reduced level of the one or more than one protein determined by comparing the level of the protein in the plant, or a tissue of the plant, with a level of the protein in a second plant, or the tissue from the second plant, that does not express the nucleic acid sequence.  
     
     
         26 . A seed comprising a silencing nucleic acid sequence and having a reduced level of one or more than one anti-nutrient compound, when compared to the level of the one or more anti-nutrient compound in a wild type seed.  
     
     
         27 . The seed of  claim 26  wherein the anti-nutrient compound is selected from the group of an intermediate of phenylpropanoid biosynthesis, a product of the phenylpropanoid pathway, sinapine, lignin, an intermediate of phytate biosynthesis, phytate.  
     
     
         28 . A method for reducing the level of one or more than one protein in a plant or a tissue within the plant comprising, 
 i) providing a plant comprising a nucleic acid sequence, the nucleic acid sequence comprising a regulatory region operatively associated with a silencing nucleotide sequence, wherein expression of the silencing nucleotide sequence reduces or eliminates the expression of two or more than two enzymes involved in the synthesis of the one or more than one protein, and    ii) expressing the silencing nucleotide sequence within the plant or a tissue within the plant, to reduce the level of the one or more than one protein in the plant or within a tissue of the plant, the reduced level of the one or more than one protein is determine by comparing the level of the one or more than one protein in the plant, or a tissue of the plant, with a level of the one ore more than one protein in a second plant, or the tissue from the second plant, that does not express the silencing nucleic acid sequence.    
     
     
         29 . The method of  claim 1 , wherein in the step of introducing, the nucleotide sequence is introduced into the plant by transformation.  
     
     
         30 . The method of  claim 1 , wherein in the step of introducing, the nucleotide sequence is introduced into the plant by crossing the plant with a second plant, the second plant comprising the nucleotide sequence.